Human RANTES (Regulated on Activation, Normal T Expressed and Secreted) is a pro-inflammatory cytokine that promotes cell accumulation and activation in chronic inflammatory diseases. It is a member of the chemokine superfamily (Schall. Cytokine. 3:165-183, 1991; Oppenheim. Ann. Rev. Immunol.9:617-648, 1991) and was originally cloned by Schall et al. (J. Immunology. 141:1018-1025,1988). hRANTES is composed of sixty eight amino acids. hRANTES shares considerable amino acid homology and indeed tertiary structural homology with hMIP-1.alpha. (LD78) and hMIP-1.beta. (ACT-2).
The following papers describe some of the functions of hRANTES, such as chemotaxis, cellular activation, recruitment of cells to sites of inflammation etc.: Taub et al., J. Leukocyte Biology 59:81-89, 1996; Ebisawa et al., J. Immunology 153:2153-2160, 1994; Rot et al., J. Exp. Med. 176:1489-1495, 1992.
RANTES expression has been demonstrated in many diseases characterised by a mononuclear cell infiltrate, suggesting that RANTES may play a role in the generation of the inflammatory infiltrate. Particular diseases or conditions in which RANTES has been implicated include: transplant rejection (particularly renal allograft rejection and lung transplantation), atherosclerosis, arthritis (particularly osteo- and rheumatoid arthritis), atopic dermatitis, airway inflammatory disorders such as Rous Sarcoma Virus-induced bronchiolitis, delayed type hypersensitivity (DTH) reactions, glomerular nephritis, asthma, endometriosis and cancers (particularly T cell lymphomas, renal cell carcinoma and Wilms' tumours (Pattison et al., Clinical Immunother. 4(1):1-8, 1995). Pattison et al., (supra) reviews the biological activities of RANTES, its expression in various disease states, and therapeutic implications. RANTES has been identified as one of the agents, termed histamine release inhibitory factors, capable of inhibiting histamine release from mast cells and basophils and has therefore been proposed as a therapeutic agent in treating allergic or chronic inflammatory diseases (WO 94/07521). RANTES plays a key role where eosinophilic tissue infiltration represents a characteristic histopathological feature. For example, eosinophil-dominated inflammation is a hallmark of asthmatic airways, and it is believed that RANTES is involved in the mechanism of eosinophil recruitment to the asthmatic bronchial mucosa (Powell et al., European Resp. Journal. 9(12):2454-2460,1996).
When agonist molecules, such as hRANTES, are administered, it may be possible to desensitise the cells and paradoxically achieve an apparent antagonist effect. This phenomenon has been demonstrated with the related chemokine IL-8 (Hechtman et al., J. Immunol 147:883-892, 1991; Smith et al., Immunology 78:491-497, 1993). Alam et al., (Am. J. Respir. Cell Mol. Biol. 7:427-433, 1992) also demonstrated that both IL-8 and RANTES inhibited monocyte chemoattractant peptide-1- and histamine releasing factor-induced histamine release from basophils in a dose-dependent fashion at concentrations of 10.sup.-9 to 10.sup.-7 M.
The following papers describe some of the disease states in which hRANTES is implicated and represent potential targets for desensitisation therapy: Kurashima et al., J. Leukocyte Biology 59:313-316, 1996, for asthma; Robinson et al., Clin. Exp. Immuno 101:398-407, 1995, for rheumatoid arthritis; Godiska et al., J. Neuroimmunology 58:167-176, 1995, for multiple sclerosis and; Khorram et al., Amer. J. Obs. Gyn. 169:1545-1549, 1993, for endometriosis.
Human RANTES along with several other chemotactic cytokine molecules (chemokines) are known to be able to suppress HIV infection in in vitro systems (Canque and Gluckman. Blood 84(10) Suppl 1: p480a, Abstract No.1907, 1994; Cocchi et al., Science 270:1811-1815, 1995; Paxton et al., Nat Med 2:412-417, 1995). The chemokines described to date which have this HIV inhibitory activity are hMIP-1.alpha., hMIP-1 .beta. and hRANTES. hMIP-1.alpha. and hMIP-1.beta. are also known as LD78 and ACT-2 respectively.
The mechanism whereby chemokines and hRANTES in particular suppress the infectivity of HIV has not been described in detail. At a gross level, it would appear that hRANTES binds to G-protein coupled receptors (GPCRs) that act as co-factors necessary for HIV infectivity and effectively block infectivity by doing so. Four chemokine receptors: Fusin (CXCR4), CCR2b, CCR3 and CCR5 have been shown to act as HIV-1 co-factor receptors (Deng et al., Nature 381:661-666; Dragic et al., Nature 381:667-674). Arenzana-Seisdedos et al. (Nature. 383:400, 1996) recently reported that an 8 amino acid N-terminally truncated analogue of human RANTES which has lost the normal RANTES chemotactic and leukocyte-activating properties but still retained high affinity for chemokine receptors, was still capable of inhibiting HIV infection, indicating that receptor signalling and cell activation is probably not required for the anti-HIV effect of RANTES, and that mere blockade of the receptor may be sufficient.
Schmidtmayerova et al. (Nature. 382:767, 1996) reported that in sharp contrast to the observed antiviral properties on T-cells, the three chemokines hMIP-1.alpha., hMIP-1.beta. and hRANTES (at 500 ng/ml) apparently stimulated HIV-1 replication in macrophages. This effect was reported to be dose dependent (although no data was presented). They conclude that administration of high levels of .beta.-chemokines could actually be harmful by enhancing HIV-1 replication in macrophages and/or intensifying virus-induced inflammation.
In contrast to the majority of human chemokines, hMIP-1.alpha., hMIP-1.beta. and hRANTES have the tendency to aggregate into large multimeric complexes in physiological solution. Such multimeric complexes are large, estimated at many times (&gt;12.times.) the mass of the monomeric protein as determined by sedimentation equilibration analyti ultracentrifugation (AUC).
Although hRANTES, hMIP-1.alpha. and hMIP-1.beta. have been implicated as possible agents for use in HIV therapy and other disease therapies, they are difficult to manufacture because of their tendency to aggregate into large multimeric complexes in physiological solution. They are also difficult to formulate due to the heterogeneity within their solutions. Disaggregated mutants of these chemokine molecules would have the benefits of easier manufacture, more defined solution characteristics and improved, reproducible formulation. These benefits are therefore particularly relevant to the clinical use of these chemokines.
WO-A-9313206 teaches the construction of disaggregated mutants of stem cell inhibitory proteins such as LD78.
An additional problem associated with the clinical administration of RANTES relates to its pro-inflammatory properties. When administered at high concentrations such as at an injection site, inappropriate immune stimulation and inflammation can occur. A single intradermal injection of RANTES into dog skin resulted in a large, dose-dependent, eosinophil and macrophage-rich inflammatory site within 4 hours, providing evidence that RANTES has significant pro-inflammatory activity (Meurer et al. J. Exp. Med. 178:1913-1921, 1993). The inflammatory response was mild at low doses (.ltoreq.0.5 .mu.M hRANTES at the injection site) but led to full dermal thickening at higher doses (.gtoreq.1 .mu.M hRANTES at the injection site). In the context of using RANTES for therapeutic treatments, such inflammatory responses could amount to serious undesirable side-effects There is a need therefore, for non- or less-inflammatory RANTES analogues, and particularly those that retain the native agonist properties.
hRANTES has been shown to act via two independent signal transduction pathways in T-cells (Bacon et al., Science 269:1727, 1995). The high affinity G-protein coupled receptor (GPCR) signalling pathway is typical of chemokines and acts at relatively low agonist concentrations eg. 50 nM hRANTES. The high affinity GPCR mediated hRANTES signalling provides all of the "usual" activities of hRANTES. These are the activities which may be demonstrated at low concentrations of hRANTES (&lt;100 nM) and include chemotaxis of leucocytes, calcium mobilisation via the GPCR and suppression of HIV infection, although the latter may only require receptor binding and not signalling. The low affinity signalling pathway is via the tyrosine kinase (TK) activity of the T-cell receptor complex and is observed only at higher agonist concentrations, &gt;&gt;100 nM, eg 1 .mu.M hRANTES. Activation of the T-cell receptor complex leads to proliferation of T-cells, induction of interleukin-2 (IL-2) expression (a pro-inflammatory cytokine) and induction of IL-2 receptor expression (Bacon et al., 1995 supra). These hRANTES-induced events are antigen independent and represent inappropriate immune stimulation. The low affinity T-cell receptor mediated signalling which causes T-cell activation and calcium mobilisation via the T-cell receptor TK is a pro-inflammatory event which may be linked to chronic inflammatory conditions and is therefore not desirable.
Bacon et al. further characterised the effects of 1 .mu.M hRANTES on T-cell activation in subsequent publications (Bacon et al., J. Exp. Med 184:873-882, 1996, and Szabo et al., Eur. J. Immunol. 27:1061-1068, 1997). In these studies they showed that hRANTES induced the phosphorylation of many proteins which play a central role in the development of T-cell focal adhesions and T-cell activation. They also found that hRANTES induced the expression of cell surface adhesion molecules such as CD44, CD50 and CD28 via the tyrosine kinase signal transduction pathway. They summarised that hRANTES was a potent immune modulator, distinct from antigen, which was able to activate T-cells resulting in their proliferation and homotypic adhesion. They speculated that activation of lymphocytes by hRANTES may be an important factor in pathologies characterised by high concentrations of RANTES but lacking other obvious antigenic stimulation.
In Bacon et al., (1996) ibid and in Szabo et al., ibid., the tendency of hRANTES to aggregate was noted but considered irrelevant to T-cell activation and homotypic aggregation. Indeed the authors implied that only monomeric or dimeric hRANTES was active and that aggregation would reduce the concentration of hRANTES active on T-cells and lymphocytes.
In addition to T-cell/lymphocyte activation and homotypic aggregation, hRANTES also induces the expression of cell-surface adhesion molecules such as CD11b (Conklyn et al., Cytokine 8:762-766, 1996). Conklyn et al. showed that hRANTES induced a dose-dependent elevation in CD11b by monocytes, neutrophils and eosinophils in human whole blood. The elevation of CD11b was observed at a threshold of 10.sup.-8 M to 10.sup.-7 M hRANTES, peaking at 10.sup.-6 M hRANTES in their assay system. These doses of hRANTES correlate with the findings of Bacon et al., (1 995; ibid) and Szabo et al., (ibid).
The study of chemokine-receptor interactions and in particular hRANTES-receptor interactions is in its infancy. However, four human cellular receptors, CCR1, CCR4, CCR5 and Duffy and one viral receptor, US28, which interact with hRANTES with relatively high affinity have been studied in some detail.
Chemokine-receptor interactions have generally been studied in two ways; 1) direct receptor binding via competitive displacement studies, and 2) signal transduction studies, with the best studies using a combination of the two approaches.
When chemokine-receptor interactions have been studied, both homologous and heterologous competition experiments have been used. A radiolabelled ligand can be used to investigate the relative affinity of several cold competing chemokines in a heterologous experiment. A preferred strategy is to perform homologous competition experiments where the radiolabelled and cold chemokines are the same.
The three chemokines MIP-1.alpha., MIP-1.beta. and RANTES are the primary ligands for the CCR5 receptor, and it is these chemokines which inhibit HIV-1 infection via the receptor (Cocchi et al., 1995, ibid). Of these, RANTES is the most potent inhibitor of HIV-1 infectivity in vitro. This observation alone suggests the importance of being able to investigate the homologous interaction of RANTES on the CCR5 receptor. Combadiere et al., (J. Leukocyte Biol. 60:147-152, 1996) who were one of the first to identify the monocytotropic HIV-1 co-factor receptor, CCR5, claim however, that it is not possible to demonstrate convincing homologous hRANTES receptor binding to CCR5. Indeed, it is recognised in the art that there are great difficulties with homologous hRANTES displacement studies. Ben-Baruch et al. (J. Biol. Chem. 270:22123-22128, 1995) described these difficulties using cell lines transfected with the CCR1 receptor. While the cell lines were suitable for MlP-1.alpha. homologous displacement studies, homologous hRANTES displacement was incomplete and biphasic. More than 30% of total counts bound could not be displaced by excess cold hRANTES and less than 70% of the total counts bound were specifically and reversibly bound to the receptor. They suggested that the problems were associated with the aggregation of hRANTES. Similar results with CCR1 receptors have been reported by others (Proudfoot et al., J. Biol. Chem. 271: 2599-2603, 1996). Similar incomplete displacement (&gt;30% of bound counts were not displaceable) have also been found with homologous hRANTES-CCR4 binding experiments (Hoogewerf et al., (Biochem. Biophys. Res. Comm. 218:337-343, 1996), and with studies using the Duffy receptor (Neote et al., Blood 84:44-52, 1994).
The art recognises therefore, that there are problems with hRANTES homologous receptor binding studies. Use of a disaggregated RANTES molecule as a ligand in receptor binding studies may alleviate these problems.